Bacterial endotoxins, also known as pyrogens, are the fever-producing byproducts of Gram negative bacteria and can be dangerous or even deadly to humans. Symptoms of infection may range from fever, in mild cases, to death. In order to promptly initiate proper medical treatment, it is important to identify, as early as possible, the presence of an endotoxin and, if possible, the concentration of the endotoxin in the subject of interest. Similarly, the U.S. Food and Drug Administration (USFDA) requires certain manufacturers to establish that their products, for example, parenteral drugs and medical devices, are free of detectable levels of Gram negative bacterial endotoxin.
To this end, a variety of methods have been developed for use in the detection of bacterial endotoxins. A currently preferred method involves the use of amebocyte lysate (AL) produced from the hemolymph of a horseshoe crab, for example, a horseshoe crab selected from the group consisting of Limulus polyphemus, Tachpleus gigas, Tachypleus tridentatus, and Carcinoscorpius rotundicauda. Amebocyte lysates produced from Limulus, Tachpleus, and Carcinoscorpius maybe referred to as LAL, TAL, and CAL, respectively.
Presently, LAL is employed in bacterial endotoxin assays of choice because of its sensitivity, specificity and relative ease for avoiding interference by other components that may be present in a sample of interest. LAL, when combined with a sample containing bacterial endotoxin, reacts with the endotoxin to produce a product, for example, a gel or chromogenic product, that can be detected, for example, either visually or by the use of an optical detector.
The endotoxin-mediated activation of LAL is well understood and has been thoroughly documented in the art. See, for example, Levin et al. (1968) Thromb. Diath. Haemorrh. 19: 186, Nakamura et al. (1986) Eur. J. Biochem. 154: 511, Muta et al. (1987) J. Biochem. 101: 1321, and Ho et al. (1993) Biochem. & Mol. Biol. Int. 29: 687. When bacterial endotoxin is contacted with LAL, the endotoxin initiates a series of enzymatic reactions, referred to in the art as the Factor C pathway, that involve at least three serine protease zymogens called Factor C, Factor B and pro-clotting enzyme (see FIG. 1). Briefly, upon exposure to endotoxin, the endotoxin-sensitive factor, Factor C is activated. Activated Factor C thereafter hydrolyses and activates Factor B, whereupon activated Factor B activates proclotting enzyme to produce clotting enzyme. The clotting enzyme thereafter hydrolyzes specific sites, for example, Arg.sup.18 -Thr.sup.19 and Arg.sup.46 -Gly.sup.47 of coagulogen, an invertebrate, fibrinogen-like clottable protein, to produce a coagulin gel. See, for example, U.S. Pat. No. 5,605,806.
Although the clotting cascade of LAL initially was considered specific for endotoxin, it was later discovered that (1.fwdarw.3)-B-D glucans also activate the clotting cascade of LAL through a unique enzymatic pathway, referred to in the art as the Factor G pathway (see FIG. 1). Upon exposure to (1.fwdarw.3)-B-D glucan, Factor G is activated to produce activated Factor G. Activated Factor G thereafter converts the proclotting enzyme into clotting enzyme, whereupon the clotting enzyme converts coagulogen into coagulin, similar to the case with endotoxin. Accordingly, the coagulation system of LAL, like the mammalian blood coagulation system, consists of at least two coagulation cascades which include an endotoxin-mediated pathway (the Factor C pathway), and a (1.fwdarw.3)-B-D glucan-mediated pathway (the Factor G pathway). See, for example, Morita et al. (1981) FEBS Lett. 129: 318-321 and Iwanaga et aL (1986) J. Protein Chem. 5: 255-268.
In view of the Factor C and Factor G pathways of LAL, the detection of bacterial endotoxin in a sample can, under certain circumstances, become ambiguous. As a result, attempts have been made to increase the specificity of LAL for endotoxin, i.e., to produce an endotoxin-specific amebocyte lysate preparation.
In one approach, polysaccharide based Factor G inhibitors are combined with amebocyte lysate to reduce or eliminate clotting induced by (1.fwdarw.3)-B-D glucan present in the biological sample, i.e., inhibit the Factor G cascade. See, for example, U.S. Pat. Nos.: 5,155,032; 5,179,006; 5,318,893; 5,474,984; and 5,641,643.
In an alternative approach, several groups have attempted to remove Factor G from LAL thereby to produce a Factor G depleted amebocyte lysate that is insensitive to (1.fwdarw.3)-B-D glucan. For example, Obayashi et al. (1985) Clin. Chim. Acta 149:55-65 disclose a method for fractionating coagulation enzymes in LAL and then recombining only those factors involved in the endotoxin induced coagulation cascade (i.e., the Factor C cascade) to produce a Factor G depleted amebocyte lysate. The resulting lysate, however, may not only lack Factor G but also other components required for a complete Factor C cascade. The reconstituted lysate produced by this procedure, apparently does not produce a natural coagulin type clot and can be used only with synthetic chromogenic substrates.
U.S. Pat. No. 5,401,647 discloses a method for removing Factor G from LAL by combining LAL with (1.fwdarw.3)-B-D glucan immobilized on an insoluble carrier. Once bound to the carrier via the (1.fwdarw.3)-B-D glucan moiety, the Factor G can thereafter be removed from the LAL to produce a Factor G depleted lysate. Similarly, U.S. Pat. No. 5,605,806 discloses an immunoaffinity based method using a Factor G specific antibody to remove Factor G from LAL thereby to produce a Factor G depleted amebocyte lysate.
There still exists, however, a demand for an endotoxin-specific amebocyte lysate that can be produced economically in commercial quantities. A method for producing such an amebocyte lysate should be rapid, reproducible, inexpensive, simple to conduct, and preferably should result in an amebocyte lysate that can be used in a reliable, and quantitative determination of endotoxin in a sample of interest.